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Thursday, 7 May 2015

One of developmental biology’s most perplexing questions concerns what signals transform masses of undifferentiated cells into tremendously complex organisms, a process called ontogeny.

UB
research suggests a new paradigm, visualized

in this
diagram, for developmental global genome

programming by the nuclear FGFR1 protein.

New research by University at Buffalo scientists, published last week in PLOS ONE, provides evidence that it all begins with a single “master” growth factor receptor that regulates the entire genome.

“The finding provides a new level of understanding of the fundamental aspects of how organisms develop,” says senior author Michal K. Stachowiak, PhD, professor in the Department of Pathology and Anatomical Sciences in the UB School of Medicine and Biomedical Sciences and senior author. He also directs the Stem Cell Engraftment and In Vivo Analysis Facility and the Stem Cell Culture and Training Facility at the Western New York Stem Cell Culture and Analysis Center at UB.

“Our research shows how a single growth factor receptor protein moves directly to the nucleus in order to program the entire genome,” he said.

Michal
Stachowiak, PhD, Professor, Department

of Pathology and Anatomical Sciences.

The research challenges a long-held supposition in biology that specific types of growth factors only functioned at a cell’s surface. For two decades, Stachowiak’s team has been intrigued by the possibility that growth factors function from within the nucleus, a point, he says, this current paper finally proves.

A more advanced understanding of how organisms form, based on this work, has the potential to significantly enhance the understanding and treatment of cancers, which result from uncontrolled development as well as congenital diseases, the researchers say. The new research also will contribute to the understanding of how stem cells work.

This work was conducted on mouse embryonic stem cells, not human cells.

Organizing ‘this cacophony of genes’

“We’ve known that the human body has almost 30,000 genes that must be controlled by thousands of transcription factors that bind to those genes,” Stachowiak said, “yet we didn’t understand how the activities of genes were coordinated so that they properly develop into an organism.”

“Now we think we have discovered what may be the most important player, which organizes this cacophony of genes into a symphony of biological development with logical pathways and circuits,” he said.

At the centre of the discovery is a single protein called nuclear Fibroblast Growth Factor Receptor 1 (nFGFR1).

“FGFR1 occupies a position at the top of the gene hierarchy that directs the development of multicellular animals,” said Stachowiak.

The FGFR1 gene is known to govern gastrulation, occurring in early development, where the three-layered embryonic structure forms. It also plays a major role in the development of the central and peripheral nervous systems and the development of the body’s major systems, including muscles and bones.

To study how nuclear FGFR1 worked, the UB team used genome-wide sequencing of mouse embryonic stem cells programmed to develop cells of the nervous system, with additional experiments in which nuclear FGFR1 was either introduced or blocked. The researchers found that the protein was responsible, either alone or with so-called partner nuclear receptors, for ensuring that embryonic stem cells develop into differentiated cells. By targeting thousands of genes, it controls the development of the major points of growth in the body (known as axes) as well as neuronal and muscle development.

The research shows that nuclear FGFR1 binds to promoters of genes that encode transcription factors, the proteins that control which genes are turned on or off in the genome.

“We found that this protein works as a kind of ‘orchestration factor,’ preferably targeting certain gene promoters and enhancers. The idea that a single protein could bind thousands of genes and then organize them into a hierarchy, that was unknown,” Stachowiak said.

“Nobody predicted it.”

Sequencing advances

The discovery that a single protein can exert such a global genomic function stems from recent advances in DNA sequencing technologies, which allow for the sequencing of a complex genome in just hours.

In the UB research, the DNA sequencing data were processed by the supercomputer at the university’s Center for Computational Research (CCR). Stachowiak and his colleagues then spent weeks aligning these data to the genome and conducting further analyses.

“We imposed nuclear FGFR1 on every little corner of genome,” he said.

“The computer spit out which genes are affected by nuclear FGFR1: it was an enormously complex network of genome activity.”

They found that the protein binds to genes that make neurons and muscles as well as to an important oncogene, TP63, which is involved in a number of common cancers.

Other studies in Stachowiak’s laboratory demonstrate that these interactions also take place in the human genome, controlling function and possibly underlying diseases like schizophrenia. Targeting of the nuclear FGFR1 allows for the reactivation of neural development in the adult brain in preclinical studies and thus, Stachowiak says, may offer unprecedented opportunity for regenerative medicine. Nuclear accumulation of nuclear FGFR1 may be altered in some cancer cells, and thus could become a focus in cancer therapy, he added.

“This seminal discovery lends new perspectives to the origin, nature and treatment of a variety of human disease,” Stachowiak concluded.

Scientists at the Salk Institute have discovered a novel type of pluripotent stem cell – cells capable of developing into any type of tissue – whose identity is tied to their location in a developing embryo. This contrasts with stem cells traditionally used in scientific study, which are characterized by their time-related stage of development.

In the paper, published May 6, 2015 in Nature, the scientists report using these new stem cells to develop the first reliable method for integrating human stem cells into nonviable mouse embryos in a laboratory dish in such a way that the human cells began to differentiate into early-stage tissues.

“The region-specific cells we found could provide tremendous advantages in the laboratory to study development, evolution and disease, and may offer avenues for generating novel therapies,” says Salk Professor Juan Carlos Izpisua Belmonte, senior author of the paper and holder of Salk’s Roger Guillemin Chair.

The researchers dubbed this new class of cells “region-selective pluripotent stem cells,” or rsPSCs for short. The rsPSCs were easier to grow in the laboratory than conventional human pluripotent stem cells and offered advantages for large-scale production and gene editing (altering a cell’s DNA), both desirable features for cell replacement therapies.

Juan Carlos Izpisua Belmonte and Jun Wu

Credit: Courtesy of the Salk Institute for

Biological Studies.

To produce the cells, the Salk scientists developed a combination of chemical signals that directed human stem cells in a laboratory dish to become spatially oriented.

They then inserted the spatially oriented human stem cells (human rsPSCs) into specific regions of partially dissected mouse embryos and cultured them in a dish for 36 hours. Separately, they also inserted human stem cells cultured using conventional methods, so that they could compare existing techniques to their new technique.

While the human stem cells derived through conventional methods failed to integrate into the modified embryos, the human rsPSCs began to develop into early stage tissues. The cells in this region of an early embryo undergo dynamic changes to give rise to all cells, tissues and organs of the body. Indeed the human rsPSCs began the process of differentiating into the three major cell layers in early development, known as ectoderm, mesoderm and endoderm. The Salk researchers stopped the cells from differentiating further, but each germ layer was theoretically capable of giving rise to specific tissues and organs.

The new stem cell (green), developed at the
Salk

Institute, holds promise for one day growing

replacement functional cells and tissues. Credit:

Courtesy of the Salk Institute for Biological

Studies.

Collaborating with the labs of Salk Professors Joseph Ecker and Alan Saghatelian, the Izpisua Belmonte team performed extensive characterization of the new cells and found rsPSCs showed distinct molecular and metabolic characteristics as well as novel epigenetic signatures – that is, patterns of chemical modifications to DNA that control which genes are turned on or off without changing the DNA sequence.

“The region selective-state of these stem cells is entirely novel for laboratory-cultured stem cells and offers important insight into how human stem cells might be differentiated into derivatives that give rise to a wide range of tissues and organs,” says Jun Wu, a postdoctoral researcher in Izpisua Belmonte’s lab and first author of the new paper.

“Not only do we need to consider the timing, but also the spatial characteristics of the stem cells. Understanding both aspects of a stem cell’s identity could be crucial to generate functional and mature cell types for regenerative medicine.”

Wednesday, 6 May 2015

Scientists at the University of Copenhagen have identified one mechanism that explains how some stem cells choose to become a given cell type: the cells combine specific sets of proteins at precise positions along the DNA. When these particular groups of proteins are combined, the gates are opened so that certain groups of genes can now be used, giving the cells a new identity.

Scientists have now identified one of these combinations, which drive the cells along the path that allow them to become organs such as liver and pancreas. This latest research could lead scientists to a better understanding on how to generate insulin-producing cells in the laboratory to use as therapy for Type I diabetes. The work has just been published in the journal Cell Stem Cell.

Specificity – choosing the combinations

Scientists working under the leadership of Henrik Semb from the DanStem Center at the University of Copenhagen have explained how the acquisition of a new cell identity is achieved; cells respond to information from their surroundings, in turn activating a specific combination of proteins at certain places on the DNA, to turn on a genetic programme.

Stem
cells.

“We added one particular chemical compound to the culture media to promote the generation of new cell types. The information transmitted by this compound is deciphered only by a small number of proteins. We then looked all along the cell’s DNA to find the positions of the proteins that were activated by the compound. We repeated the experiment using additional compounds, to get an idea of how specific the responses were and to categorize the genes that the cells decided to use when being directed toward different cellular fates,” says Assistant Professor Karen Schachter.

Getting the identity right

The work in the field of human pluripotent stem cell research has concentrated on finding the correct combination of drugs or chemical compounds that can be used to drive the cells into specific cell types in the culture dish.

“There is however a lack of understanding of how these compounds activates the genes that give the cells unique identities, which has resulted in a lack of reproducibility of the methods used by different labs. As a comparison; if you use a pre-mixed powder to bake a cake you will face problems if you run out on an important ingredient and do not know how to replace its action. We believe that our study provides useful information that will help us to understand the recipe better, so that we can generate functional cells in a more controlled manner,” adds Post doc Nina Funa.

There is already a lot of focus in the stem cell community to generate cells in the laboratory to use as therapy, so the scientists at DanStem want to emphasize the importance of continuing doing this important basic research work.

“Our ultimate aim is to understand how stem cells make choices, which will also help improve the quality of the work that will put stem cells into therapeutic use,” concludes Funa.